The knowledge about the human genome in the last decade has helped in our understanding of the causes and molecular mechanisms for manifestation of various diseases. It is a well-accepted fact that the treatment for several monogenic and acquired genetic disorders as well as complex diseases like cancer can be fully or partially achieved by different types of gene therapy approaches [Rubanyi G M, 2001; Howard K A, 2009; Petros R A et. al., 2010]. Adopting such approaches is limited by the availability of safe and efficient vectors which can deliver the desired nucleic acid for gene expression/suppression to affected cells/tissues in the body. Viral gene delivery systems have been largely explored and account for more than 70% of clinical trials but their practical applicability to the clinics is still questionable due the associated risks like insertional mutagenesis and immune activation [Mark A K et. al., 2003]. Lipid and polymer based non-viral vectors provide a safer alternative and can also carry large cargo molecules unlike viral vectors but are of limited use because of their inability to overcome all the barriers encountered during in vivo delivery of nucleic acids and long term toxicity and biocompatibility issues. Additionally their synthesis demands specialized expertise and can often result in limited product yield posing economic constraints [Mintzer M A et. al., 2009; Remaut K et. al., 2007].
A more recent rational approach is the integration of different non-viral vectors such that they can complement each other's functions and systematically cross the hurdles encountered during in vivo gene delivery. Peptides are the best choice in this regard since different peptides possess the inherent ability to cross various cellular barriers like plasma, endosomal and nuclear membranes [Remaut K et. al., 2007; Mintzer M A et. al., 2009]. Peptides can also package different forms and sizes of nucleic acids to form nanocomplexes, and serve as cellular targeting ligands. Other advantages such as ease of synthesis and chemical modifications, relatively low toxicity and immunogenicity make peptides an attractive class of non-viral nucleic acid delivery agents [Vazquez E et. al., 2008; Martin M E et. al., 2007; Morris M C et. al., 2000; Hart S L et. al., 2010; Mann A et. al., 2008]. Peptides have been described in the literature for delivery of biomolecules like DNA and siRNA. Of these, lysine and arginine-rich peptides are the most promising vectors for plasmid DNA delivery since they can efficiently condense DNA and form nanocomplexes to prevent enzymatic degradation inside the cell [Mann A et. al., 2007]. In addition, arginine peptide show strong cellular uptake [Biessen E A L et. al., 2004; Wang S et. al., 2008; Harashima H et. al., 2009; Kim Y H et. al., 2009]. Arginine homopeptides possess better DNA condensation and release balance than their cognate lysine variants and thus are more efficient. However, the efficiency of delivery of a large cargo like plasmid DNA in the form of a nanocomplex by lysine and arginine homopeptides is rather low because of their inability to overcome the endosomal barrier effectively. Modification of lysine peptides in these nanocomplexes with histidines in linear as well as branched form have been shown to add endosomal escape property to the DNA condensing system [Kichler et. al., 2007]. However, such systems involve complicated design and synthesis steps and are not very efficient.
There is a need to design nanocomplexes containing cationic peptide which are able to overcome all the barriers for intracellular entry by showing high cellular uptake, efficient endosomal escape and high transfection efficiency with low toxicity.
In the present work we have explored various arginine-histidine-cysteine combinations and have developed nanocomplexes containing two novel cationic peptides with combinations of 9 arginine residues, 7 histidine residues and 2 cysteine residues at both ends that can efficiently overcome endosomal barrier but retain their DNA-condensation and release balance. Arginines have been used as condensing moieties, histidines are involved in efficient endosomal escape and the addition of cysteines further enhances the DNA condensation and release balance possibly through formation of reducible cross-linkages. These nanocomplexes show remarkable efficiency of plasmid DNA delivery comparable to commercially available transfection reagents in various cell lines including the ones which are tough to transfect. These particular nanocomplexes comprising linear arginine-histidine-cysteine sequences of short length (as described in the sequence listings) with high DNA delivery efficiency and low toxicity are completely unique.
Lysine/Arginine-histidine-cysteine nanocomplex systems listed in the literature are either (a) in the form of reducible polycations (RPCs) where the peptide has been polymerized before making a nanocomplex with the DNA or (b) the peptide-DNA nanocomplex is oxidized [Wang S et. al., 2008; Kim Y H et. al., 2009; Kim Y H et. al., 2010]. Such oxidation steps are not required in our nanocomplex system. This is an obvious advantage of our system over existing ones.